Chemical gradients generated by the release and diffusion of soluble factors mediate many complex processes in biology. A chemical gradient provides not only information about the existence of a particular signal, but also provides spatial information. For example, a motile cell moving through a gradient in a direction of increasing concentration will eventually find the source of the chemical signal. This process, i.e., the migration of cells towards or away from a source of a chemical signal (chemotaxis), plays a key role in cancer spreading, wound healing, and morphogenesis in the case of eukaryotic cells; and animal infection, plant infection, and carbon cycling in the ocean in the case of prokaryotic cells.
In-vitro models of biochemical gradients have been used to validate cellular models by assessing cellular response to specific soluble signals. Early approaches to study chemotaxis provided a wealth of useful information despite the limitations of having to deal with microscale phenomena using macroscale tools. Microfabrication techniques provide tools that allow for exquisite control over microscale liquid interfaces and in particular, control of chemical gradients. For example, Jeong et al. showed that in a microchannel, under laminar flow and high Peclect number (ratio of convection to diffusion), a cross-sectional concentration profile generated upstream is preserved downstream as it flows over a cell population under study, see N. L. Jeon, H. Baskaran, S. K. W. Dertinger, G. M. Whitesides, L. Van de Water and M. Toner, “Neutrophil Chemotaxis in Linear and Complex Gradients of Interleukin-8 Formed in a Microfabricated Device,” Nat Biotechnol, Vol. 20, pp. 826-830 (August 2002). Subsequent monitoring of cell motility was correlated with the gradient profile to infer chemotactic behavior.
Due to the relative simplicity of this approach, gradients generated using laminar flow have been used extensively to study chemotaxis of adherent cells. However, a disadvantage of this practice is that cells may be exposed to convective flows which cells may not experience in-vivo. The side effects of such convective flows may include for example, shear stress, the removal of autocrine and paracrine signals secreted by cells, bias on the direction of cellular migration and asymmetrical mass transport, all of which may alter cellular response. In some cases, research has been devoted, if not to eliminate these adverse effects, at least to minimize them. In general, fluid flow is not well suited to analyze chemotaxis of non-adherent cells because instead of flushing soluble signals over the cells, it may flush both signals and cells. An exception is when the object of study is the competition between bacterial migration towards nutrients, against external adverse convection of turbulences.
Recent attempts to generate diffusive gradients in the absence of convective flows have yielded important but partial progress. These approaches include (i) using membranes to restrict flow while allowing diffusion of soluble species, (ii) using valves to bring liquid plugs in contact allowing free diffusion between them; and (iii) different configurations for balancing pressures on both sides of a microchannel. Although each of these elegant approaches has provided valuable insight into chemotaxis, all of them have limitations. Typically they (i) allow only for the diffusion between two solutions (one dimensional); (ii) are static; i.e., once the diffusive interface is established, it can only be modified using convective flows that inevitably disrupt the diffusion profiles; (iii) can operate only for short periods of time; or (iv) have difficult accessibility, in that cells need to be introduced in the chambers before the gradients are generated. The transient development of a gradient can be long and influence cellular response in ways that may not be easily deconvolved.
Prior artisans have devices and strategies for forming gradients. However, these previous efforts have suffered from one or more of the previously noted disadvantages. For example, US Patent Publication 2007/0253868 to Beebe et al. is directed to a microfluidic platform and method for generating a gradient. However, those various devices all utilize one or more membranes to prevent convective flow into a chamber region. As noted, such membranes interfere with extent and rate of diffusion of agents into the chamber. U.S. Pat. No. 7,306,672 to Hansen et al. provides a detailed description of microfluidic devices that provide for diffusion into a chamber or region of interest. However, the devices and systems described by Hansen et al. utilize flow-blocking valves in one or more microfluidic chambers. Upon establishment of static liquids on both sides of a valve, the valve is then opened whereby diffusion can then occur across the resulting liquid interface. Although satisfactory in certain respects, using one or more valves to selectively create such a liquid interface introduces additional variables which may deleteriously affect gradient development and maintenance. Furthermore, requiring the use of moving components in a microfluidic system increases complexity and cost of the system. Additionally, using flow-blocking valves precludes administration of one or more agents from a source into a chamber or other region of interest, while the valve(s) are closed. Moreover, pressure differences between liquids on opposite sides of the valve and opening or closing operations of the valve itself can introduce or generate undesirable convective flows.
Additionally, gels have also been used to prevent convective flows and simultaneously to grow cells in three dimensional scaffolds and other microfluidic devices. However, gels as a result of their high viscosity, are often not appropriate for many investigations.
In view of these various efforts and their limitations, a need exists for a strategy by which gradients may be readily formed and maintained without the previously noted deleterious consequences associated with currently known microfluidic devices and the convective flows that inevitably result therein. Specifically, it would be desirable to provide a process and device for establishing and maintaining one or more gradients exclusively by diffusion and without the use of membranes, flow-blocking valves, or gels.